An Epitope-Specific LGI1-Autoantibody Enhances Neuronal Excitability by Modulating Kv1.1 Channel

Leucine-rich Glioma-Inactivated protein 1 (LGI1) is expressed in the central nervous system and its genetic loss of function is associated with epileptic disorders. Additionally, patients with LGI1-directed autoantibodies have frequent focal seizures as a key feature of their disease. LGI1 is composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. These domains are reported to interact with different members of the transsynaptic complex formed by LGI1 at excitatory synapses, including presynaptic Kv1 potassium channels. Patient-derived recombinant monoclonal antibodies (mAbs) are ideal reagents to study whether domain-specific LGI1-autoantibodies induce epileptiform activities in neurons and their downstream mechanisms. We measured the intrinsic excitability of CA3 pyramidal neurons in organotypic cultures from rat hippocampus treated with either an LRR- or an EPTP-reactive patient-derived mAb, or with IgG from control patients. We found an increase in intrinsic excitability correlated with a reduction of the sensitivity to a selective Kv1.1-channel blocker in neurons treated with the LRR mAb, but not in neurons treated with the EPTP mAb. Our findings suggest LRR mAbs are able to modulate neuronal excitability that could account for epileptiform activity observed in patients.


Introduction
Autoantibodies directed against Leucine-rich Glioma-Inactivated protein 1 (LGI1) are found in patients with limbic encephalitis (LE) who have frequent focal seizures and hippocampal atrophy as hallmarks of their disease [1][2][3]. Hyperexcitability and epileptiform activities have been recorded in the hippocampi of mice and rats treated with serum from patients with LGI1 autoantibodies, suggesting their direct pathogenicity [4,5]. LGI1 is a soluble molecule composed of a Leucine-Rich Repeat (LRR) and an Epitempin (EPTP) domain. The LRR domain is reported to mediate transsynaptic homo-oligomerization, whereas the EPTP domain allows for LGI1 to dock on its pre-and post-synaptic receptors, ADAM23 and ADAM22, respectively [6]. Serum LGI1 antibodies have been shown to target both the LRR and EPTP domains of LGI1. Sera have been shown to cause both a downregulation of two interaction partners of LGI1: the presynaptic voltage-gated potassium channel Kv1 and the postsynaptic AMPA receptors [4,5,7,8]. Furthermore, polyclonal serum antibodies can prevent the interaction of LGI1 with ADAM22 and potentiate excitatory glutamatergic synapses [7][8][9].
More recent reports aimed to better characterize the relative pathogenic roles of domain-specific antibodies by studying recombinant monoclonal LGI1 autoantibodies (mAb) derived from patient B cells [7,8]. mAbs targeting the LRR domain were shown to internalize the LGI1 protein after it docked to ADAM22/ADAM23 receptors [8]. In contrast, EPTP-mAbs operated by competing with LGI1 for binding to ADAM22 [7,8]. Both mAb before the first spike obtained under rheobase current; Figure 1B). In order to measure the effect of Kv1 channels on the ramp-and-delay phenotype, the depolarizing slope before the first action potential was measured on the 10 ms before the spike threshold [13] ( Figure  1B). The spike threshold was measured on phase plots as previously shown [14]. Both slopes and spike thresholds were measured on spikes evoked in the first 200 ms after the current pulse onset to maximize the role of Kv1.1 channels.

Figure 1. Experimental procedures. (A)
Timeline representing the patient-derived mAb incubation protocol. Hippocampal slices of rat aged of 7 to 10 post-natal days (P7-P10) were put in culture and individually incubated with either a recombinant LRR-mAb, a recombinant EPTP-mAb, or polyclonal IgGs from healthy human patient (HC IgGs) at 4 days of culture until the days of experiment at day 7 (d7) and day 8 (d8). (B) CA3 pyramidal neurons from organotypic cultures treated with the different antibodies were recorded in current-clamp mode. The number of spikes elicited by the injection of depolarizing currents with increments of 10 pA from 0 to 200 pA was measured to obtain an input-output curve. The minimal current injected to elicit at least an action potential (rheobase) was determined. The slope of the membrane potential trajectory before the action potential was quantified in a time window of 10 ms.
Sensitivity of intrinsic excitability to the selective Kv1.1-channel blocker, dendrotoxin-K (DTx-k), was determined by current-clamp recording before and after 5 min of bath application of DTx-K (100 nM). Acquisition was performed at 10 kHz with pClamp10 (Axon Instruments, Molecular Devices, San Jose, CA, USA. Data were analyzed with ClampFit (Axon Instruments), LabView (National Instruments, Austin, TX, USA) and IgorPro (Wavemetrics, Lake Oswego, OR, USA). Pooled data are presented as mean ± SE and statistical analysis was performed using the Mann-Whitney U test or Wilcoxon rank signed test.

LRR-mAbs, but Not EPTP-mAbs, Increase Intrinsic Excitability in CA3 Pyramidal Neurons
To assess the effect of the two mAbs on neuronal excitability, we performed currentclamp recordings from CA3 pyramidal neurons in organotypic cultures of rat hippocampus treated with the different Abs. Input-output curves were established for each neuron (Figure 2A). A significant decrease in the rheobase was observed after treatment with the LRR-mAb (HC IgGs: 100.1 ± 7.8 pA n = 13 vs. LRR-mAb: 75.4 ± 7.5 pA n = 14, Mann-Whitney, p = 0.035; Figure   Hippocampal slices of rat aged of 7 to 10 post-natal days (P7-P10) were put in culture and individually incubated with either a recombinant LRR-mAb, a recombinant EPTP-mAb, or polyclonal IgGs from healthy human patient (HC IgGs) at 4 days of culture until the days of experiment at day 7 (d7) and day 8 (d8). (B) CA3 pyramidal neurons from organotypic cultures treated with the different antibodies were recorded in current-clamp mode. The number of spikes elicited by the injection of depolarizing currents with increments of 10 pA from 0 to 200 pA was measured to obtain an input-output curve. The minimal current injected to elicit at least an action potential (rheobase) was determined. The slope of the membrane potential trajectory before the action potential was quantified in a time window of 10 ms.
Sensitivity of intrinsic excitability to the selective Kv1.1-channel blocker, dendrotoxin-K (DTx-k), was determined by current-clamp recording before and after 5 min of bath application of DTx-K (100 nM). Acquisition was performed at 10 kHz with pClamp10 (Axon Instruments, Molecular Devices, San Jose, CA, USA).
Data were analyzed with ClampFit (Axon Instruments), LabView (National Instruments, Austin, TX, USA) and IgorPro (Wavemetrics, Lake Oswego, OR, USA). Pooled data are presented as mean ± SE and statistical analysis was performed using the Mann-Whitney U test or Wilcoxon rank signed test.

LRR-mAbs Increase Intrinsic Excitability through the Modulation of Kv1 Channels
Based on these findings, we asked whether changes in the D-type potassium current could account for the change in intrinsic excitability by measuring the sensitivity to DTx-

Discussion
We show here that two patient-derived mAbs directed against the LRR and EPTP epitopes of LGI1 differentially perturb neuronal excitability in CA3 pyramidal neurons from rat hippocampal organotypic cultures. Compared to IgGs from control patients, LRR-mAbs but not EPTP-mAbs were found to increase neuronal intrinsic excitability. In fact, the input-output curves of LRR-mAb-treated neurons were shifted to the left due to a lower rheobase current. The ramp-and-delay phenotype that is a hallmark of CA3 pyramidal neurons disappeared in LRR-mAb-treated neurons. Finally, treatment with the LRR-mAb, but not the EPTP-mAb, prevented the effect of DTx-k, a blocker of the Kv1.1 voltage-gated potassium channel, indicating that LRR-mAb already reduced functional Kv1.1 channels.
Our results were obtained after 4-5 days of anti-LGI1 application at a concentration of 4 ng/µL. In previous studies, acute application (up to 8 h) [4] or long-lasting application of LGI1 antibodies (14 days) [5] have been shown to produce similar functional effects, suggesting that the application time is not a critical factor.
The increase in excitability observed in LRR-mAb-treated neurons is associated with a lower rheobase, a lower first spike latency, and an elevated depolarizing slope before the action potential. All these changes are mediated by Kv1.1 channels as the application of the selective Kv1.1 channel blocker, DTx-k had little effect on these parameters in LRR-mAb-treated neurons, but not in HC-and EPTP-mAb-treated neurons. The rheobase of LRR-mAb-treated neurons was slightly reduced in DTx-k, indicating that not all Kv1.1 channels are internalized by LRR-mAbs. Surprisingly, no significant change in action potential threshold was observed following treatment with LRR mAbs in our study. As the spike threshold depends on both voltage-gated sodium (Nav) and potassium channels, the lack of spike threshold change may be attributed to the homeostatic reduction of Nav channel density in LRR-mAb-treated neurons, as previously observed in over-excited neurons [15].
Our results are compatible with previous studies showing that LRR-mAbs promote LGI1-ADAM complex internalization [8], which may lead to a reduction in Kv1 channel expression at the cell membrane. This mechanism is further supported by the genetic deletion of LGI1, which decreases both Kv1.1 channel density and D-type currents by more than 50% [16]. As Kv1.1 channels are expressed at both the AIS and presynaptic terminals of CA3 pyramidal neurons [17], the putative consequence of the decrease in Kv1.1-mediated current is an elevated neuronal excitability and an elevated glutamate release, which both concur to promote synchronous epileptiform discharges. Our results were obtained in vitro, as most results describing the effects of LGI1 manipulation on neuronal excitability. It would be nevertheless important to verify that they are confirmed in an in vivo preparation [18].
Antibodies against LGI1 are known to prevent long-term synaptic potentiation (LTP) and memory formation in vivo [5,8]. Interestingly, while both LRR-mAbs and EPTP-mAbs equally prevent LTP, only LRR-mAbs impair object recognition [8], suggesting that object recognition may involve mechanisms different from LTP. However, the lack of LTP in animals treated with LGI1-Abs is difficult to interpret as epileptiform activity itself triggers synaptic potentiation that prevents further LTP induction [19]. As epileptiform activity is observed in animals treated with LGI1-Abs, there are also good reasons to believe that the lack of LTP observed in these animals is in fact due to the epilepsy-induced occlusion of LTP [20].
The molecular mechanism by which EPTP-mAbs mediate their effect is likely to occur through the prevention of LGI1 binding to its native receptors. As suggested in a recent study [21], EPTP-mAb (mAb6212) could bind LGI1 in non-neuronal complexes such as glial cells (astrocytes and oligodendrocytes) that may thus explain the lack of excitability regulation induced by this particular EPTP-mAb. Further experiments would be needed to elucidate these questions.